Substituted 2-thio-3, 5-dicyano-4-phenyl-6-aminopyridines and their use as adenosine receptor-selective ligands

ABSTRACT

Compounds of the formula (I) 
                         
a process for preparing them, and their use as medicaments are described.

The present invention relates to substituted2-thio-3,5-dicyano-4-phenyl-6-aminopyridines, to a process for preparingthem and to their use as medicaments.

Adenosine, a nucleoside consisting of adenine and D-ribose, is anendogenous factor which exhibits cell-protective activity, in particularunder cell-damaging conditions involving restricted oxygen and substratesupply, as occur, for example, in a wide variety of organs (e.g. heartand brain) in association with ischaemia.

While adenosine is formed intracellularly as an intermediate whenadenosine-5′-monophosphate (AMP) and S-adenosylhomocysteine are brokendown, it can be released from the cell and then exerts functions, bymeans of binding to specific receptors, as a hormone-like substance orneurotransmitter.

Under normoxic conditions, the concentration of free adenosine in theextracellular space is very low. However, the extracellularconcentration of adenosine increases dramatically in the affected organsunder ischaemic or hypoxic conditions. Thus, it is known, for example,that adenosine inhibits platelet aggregation and increases the flow ofblood through the coronary vessels. In addition, it affects the heartrate, the secretion of neurotransmitters and lymphocyte differentiation.

These effects of adenosine are directed towards increasing the supply ofoxygen in the affected organs and/or throttling back the metabolism ofthese organs in order, in this way, to achieve an adaptation of theorgan metabolism to the flow of blood through the organ under ischaemicor hypoxic conditions.

The effect of adenosine is mediated by way of specific receptors. Thosewhich are known to date are the subtypes A1, A2a, A2b and A3. Theeffects of these adenosine receptors are mediated intracellularly by themessenger compound cAMP. When adenosine binds to the A2a or A2breceptors, the intracellular cAMP is increased as a result of themembrane-located adenylate cyclase being activated, whereas the bindingof the adenosine to the A1 or A3 receptors brings about a decrease inthe content of intracellular cAMP as a result of the adenylate cyclasebeing inhibited.

According to the invention, those substances which are able to bindselectively to one or more of the adenosine receptor subtypes and, inthis connection, either imitate the effect of adenosine (adenosineagonists) or block its effect (adenosine antagonists) are termed“adenosine receptor-selective ligands”.

According to their receptor selectivity, adenosine receptor-selectiveligands can be subdivided into various classes, for example into ligandswhich bind selectively to the A1 or A2 adenosine receptors, and, in thelatter case, also, for example, into those which bind selectively to theA2a or A2b adenosine receptors. It is also possible for adenosinereceptor ligands to exist which bind selectively to several of theadenosine receptor subtypes, for example ligands which bind selectivelyto the A1 and A2 adenosine receptors but not to the A3 adenosinereceptors.

The abovementioned receptor selectivity can be determined, for example,by the effect of the substances on cell lines which express the relevantreceptor subtypes following stable transfection with the appropriatecDNA (in this regard, see the article M. E. Olah, H. Ren, J. Ostrowski,K. A. Jacobson, G. L. Stiles, “Cloning, expression, and characterizationof the unique bovine A1 adenosine receptor. Studies on the ligandbinding site by site-directed mutagenesis.” in J. Biol. Chem. 267 (1992)pages 10764–10770, the entire disclosure of which is hereby incorporatedby reference).

The effect of the substances on such cell lines can be determined bybiochemical measurement of the intracellular messenger compound cAMP (inthis regard, see the article K. N. Klotz, J. Hessling, J. Hegler, C.Owman, B. Kull, B. B. Fredholm, M. J. Lohse, “Comparative pharmacologyof human adenosine receptor subtypes—characterization of stablytransfected receptors in CHO cells” in Naunyn Schmiedebergs Arch.Pharmacol. 357 (1998) pages 1–9, the entire disclosure of which ishereby incorporated by reference).

The adenosine receptor ligands which are disclosed in the prior art arein the main derivatives based on natural adenosine (S.-A. Poulsen and R.J. Quinn, “Adenosine receptors: new opportunities for future drugs” inBioorganic and Medicinal Chemistry 6 (1998) pages 619–641). However,these adenosine ligands which are known from the prior art usuallysuffer from the disadvantage that they are-less active than the naturaladenosine or are only very weakly active, or not active at all,following oral administration. For this reason, they are in the mainonly used for experimental purposes.

In addition to this, WO 00/125210 discloses2-thio-3,5-dicyano-4-aryl-6-aminopyridines which are structurallysimilar to the compounds according to the invention. However, thecompounds which are described in the above publication possessdisadvantageous pharmacokinetic properties; in particular, they onlyhave low bioavailability following oral administration.

The object of the present invention is now to find or prepare compoundswhich avoid the disadvantages of the prior art, i.e. which, inparticular, possess improved bioavailability.

The present invention relates to compounds of the formula (I)

in which

-   R¹ denotes (C₁–C₄)-alkyl, (C₁–C₄)-alkoxy, or mono- or    di-(C₁–C₄)-alkylamino, and-   R² denotes pyridyl or thiazolyl, which radicals can be substituted    by halogen, amino or (C₁–C₄)-alkyl,    and their salts, hydrates, hydrates of the salts and solvates.

Depending on the substitution pattern, the compounds of the formula (I)can exist in stereoisomeric forms which either relate to each other asimage and mirror image (enantiomers) or do not relate to each other asimage and mirror image (diastereomers). The invention relates both tothe enantiomers or diastereomers and to their respective mixtures. Theracemic forms can be separated, in a known manner, in exactly the sameway as the diastereomers, into the stereoisomerically uniformconstituents. Equally, the present invention also relates to the othertautomers of the compounds of the formula (I) and their salts.

Salts of the compounds of the formula (I) can be physiologicallyharmless salts of the compounds according to the invention with mineralacids, carboxylic acids or sulphonic acids. Particular preference isgiven, for example, to salts with hydrochloric acid, hydrobromic acid,sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonicacid, toluenesulphonic acid, benzenesulphonic acid,naphthalenedisulphonic acid, trifluoroacetic acid, acetic acid,propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid,maleic acid or benzoic acid.

Salts which may also be mentioned are salts with customary bases, forexample alkali metal salts (e.g. sodium salts or potassium salts),alkaline earth metal salts (e.g. calcium salts or magnesium salts) orammonium salts which are derived from ammonia or organic amines such asdiethylamine, triethylamine, ethyldiisopropylamine, procaine,dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine ormethylpiperidine.

According to the invention, those forms of the compounds of the formula(I) which, in the solid or liquid state, form a molecule compound or acomplex by hydration with water or coordination with solvent moleculesare termed hydrates and solvates, respectively. Examples of hydrates aresesquihydrates, monohydrates, dihydrates and trihydrates. In preciselythe same way, the hydrates or solvates of salts of the compoundsaccording to the invention also come into consideration.

In addition, the invention also encompasses prodrugs of the compoundsaccording to the invention. According to the invention, those forms ofthe compounds of the formula (I) which may themselves be biologicallyactive or inactive but which can be converted (for example metabolicallyor solvolytically) into the corresponding biologically active form underphysiological conditions are termed prodrugs.

Within the context of the present invention, the substituents have,unless otherwise indicated, the following meaning:

Halogen in general represents fluorine, chlorine, bromine or iodine.Fluorine, chlorine or bromine are preferred. Fluorine or chlorine arevery particularly preferred.

(C₁–C₄)-Alkyl in general represents a straight-chain or branched alkylradical having from 1 to 4 carbon atoms. Examples which may be mentionedare: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyland tert-butyl.

(C₁–C₄)-Alkoxy in general represents a straight-chain or branched alkoxyradical having from 1 to 4 carbon atoms. Examples which may be mentionedare: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy,isobutoxy and tert-butoxy.

Mono- or di-(C₁–C₄)-alkylamino in general represents an amino grouphaving one or two identical or different straight-chain or branchedalkyl substituents which in each case possess from 1 to 4 carbon atoms.Examples which may be mentioned are: methylamino, ethylamino,n-propylamino, isopropylamino, t-butylamino, N,N-dimethylamino,N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino,N-isopropyl-N-n-propylamino and N-t-butyl-N-methylamino.

Compounds of the formula (I) are preferred

in which

-   R¹ denotes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,    isobutyl or tert-butyl, and-   R² denotes 2-pyridyl, thiazol-4-yl or thiazol-5-yl, which radicals    can be substituted by chlorine, amino or methyl,    and their salts, hydrates, hydrates of the salts and solvates.

Particular preference is given to compounds of the formula (I) in whichR¹ denotes (C₁–C₄)-alkyl and their salts, hydrates, hydrates of thesalts and solvates.

Particular preference is likewise given to compounds of the formula (I)in which R² denotes unsubstituted pyridyl and their salts, hydrates,hydrates of the salts and solvates.

Particular preference is likewise given to the compound having thefollowing formula

and its salts, hydrates, hydrates of the salts and solvates.

The present invention also relates to a process for preparing thecompounds of the formula (I) which is characterized in that

compounds of the formula (II)

in which

-   R¹ has the abovementioned meaning,    are reacted with compounds of the formula (III)    R²—CH₂—X  (III)    in which-   R² has the abovementioned meaning and X represents a suitable    leaving group, preferably halogen, in particular chlorine, bromine    or iodine, or represents mesylate, tosylate, triflate or    1-imidazolyl,    where appropriate in the presence of a base.

The above-described process can be explained, by way of example, by thefollowing formula scheme:

All organic solvents which are inert under the reaction conditions aresuitable solvents for the process according to the invention. Thesesolvents include alcohols, such as methanol, ethanol and isopropanol,ketones, such as acetone and methyl ethyl ketone, acyclic and cyclicethers, such as diethyl ether and tetrahydrofuran, esters such as ethylacetate or butyl acetate, hydrocarbons, such as benzene, xylene,toluene, hexane or cyclohexane, chlorinated hydrocarbons, such asdichloromethane, chlorobenzene or dichloroethane, or other solvents,such as dimethylformamide, acetonitrile, pyridine or dimethyl sulfoxide(DMSO). Water is likewise suitable for use as a solvent.Dimethylformamide is preferred. It is likewise possible to use mixturesof the abovementioned solvents.

The customary inorganic or organic bases are suitable for use as bases.These bases preferably include alkali metal hydroxides, such as sodiumhydroxide or potassium hydroxide, or alkali metal carbonates, such assodium carbonate or potassium carbonate, or alkali metal hydrogencarbonates, such as sodium hydrogen carbonate or potassium hydrogencarbonate, or alkali metal alkoxides, such as sodium methoxide orpotassium methoxide, sodium ethoxide or potassium ethoxide or potassiumtert-butoxide, or amides, such as sodium amide, lithiumbis(trimethylsilyl)amide or lithium diisopropylamide, or organometalliccompounds, such as butyllithium or phenyllithium, or else amines, suchas triethylamine and pyridine. The alkali metal carbonates and alkalimetal hydrogen carbonates are preferred.

In this connection, the base can be employed in a quantity of from 1 to10 mol, preferably from 1 to 5 mol, in particular from 1 to 4 mol, basedon 1 mole of the compounds of the formula (II).

In general, the reaction takes place in a temperature range from −78° C.up to +140° C., preferably in the range from −78° C. to +40° C., inparticular at room temperature.

The reaction can be carried out under normal, increased or decreasedpressure (for example in the range from 0.5 to 5 bar). In general, it iscarried out under standard pressure.

The compounds of the formula (II) are known to the skilled person or canbe prepared using customary methods which are known from the literature.

The compounds of the formula (II) can also be prepared from compounds ofthe formula (IV) by reacting them with an alkali metal sulphide. Thispreparation method can be explained, by way of example, by the followingformula scheme:

The alkali metal sulphide employed is preferably sodium sulphide, whichis employed in a quantity of from 1 to 10 mol, preferably of from 1 to 5mol, in particular of from 1 to 4 mol, based on 1 mole of the compoundsof the formula (IV).

All organic solvents which are inert under reaction conditions aresuitable for use as solvents. These include N,N-dimethylformamide,N-methylpyrrolidinone, pyridine and acetonitrile. N,N-Dimethylformamideis particularly preferred. It is likewise possible to use mixtures ofthe abovementioned solvents.

In general, the reaction takes place in a temperature range from +20° C.to +140° C., preferably in the range from +20° C. to +120° C., inparticular at from +60° C. to +100° C.

The reaction can be carried out under normal, increased or decreasedpressure (e.g. in the range from 0.5 to 5 bar). In general, it iscarried out under standard pressure.

The compounds of the formula (III) are commercially available, or areknown to the skilled person or can be prepared using customary methods.

The compounds of the formula (IV) are commercially available, or areknown to the skilled person or can be prepared using customary methods.In particular, reference may be made to the following articles, therespective contents of which are hereby incorporated by reference:

-   -   Kambe et al., Synthesis, 531–533 (1981);    -   Elnagdi et al., Z. Naturforsch.47b, 572–578 (1991).

Surprisingly, the compounds of the formula (I) exhibit a valuablepharmacological spectrum of activity, which was not predictable, and aretherefore particularly suitable for the prophylaxis and/or treatment ofdiseases.

As compared with the prior art, the compounds of the formula (I)according to the invention possess superior pharmacokinetic properties,in particular a superior bioavailability following oral administration.

The compounds of the formula (I) are suitable, either alone or incombination with one or more different active compounds, for theprophylaxis and/or treatment of various diseases such as, for example,diseases of the cardiovascular system, in particular. Suitable activecompounds for use in combination are, in particular, active compoundsfor treating coronary heart diseases, for example nitrates,betablockers, calcium antagonists and diuretics, in particular.

Within the meaning of the present invention, cardiovascular diseases areto be understood, for example, as being the following diseases, inparticular: coronary heart disease, hypertension (high blood pressure),restenosis, such as restenosis following balloon dilatation ofperipheral blood vessels, arteriosclerosis, tachycardias, arrhythmias,peripheral and cardiac vascular diseases, stable and unstable anginapectoris and atrial fibrillation.

The compounds of the formula (I) are furthermore suitable, for example,for reducing the myocardial region affected by an infarction, inparticular.

In addition, the compounds of the formula (I) are suitable, for exampleand in particular, for the prophylaxis and/or treatment ofthromboembolic diseases and ischaemias, such as myocardial infarction,cerebral stroke and transitory ischaemic attacks.

Examples of other indication areas for which the compounds of theformula (I) are suitable are, in particular, the prophylaxis and/ortreatment of diseases of the urogenital region, such as irritablebladder, erectile dysfunction and female sexual dysfunction, and, inaddition, however, also the prophylaxis and/or treatment of inflammatorydiseases, such as asthma and inflammatory dermatoses, ofneuroinflammatory diseases of the central nervous system, such asconditions occurring after cerebral infarction, and of Alzheimer'sdisease, and, furthermore, also of neurodegenerative diseases, and alsoof pain and cancer.

An example of another indication area is, in particular, the prophylaxisand/or treatment of diseases of the airways, such as asthma, chronicbronchitis, pulmonary emphysema, bronchiectases, cystic fibrosis(mucoviscidosis) and pulmonary hypertension.

In addition, the compounds of the formula (I) are also suitable, forexample and in particular, for the prophylaxis and/or treatment of liverfibrosis and liver cirrhosis.

Finally, the compounds of the formula (I) are also suitable, for exampleand in particular, for the prophylaxis and/or treatment of diabetes, inparticular diabetes mellitus.

The present invention also relates to the use of the compounds of theformula (I) for producing medicaments for the prophylaxis and/ortreatment of the above-mentioned syndromes.

The present invention furthermore relates to a process for theprophylaxis and/or treatment of the abovementioned syndromes using thecompounds of the formula (I).

The pharmaceutical activity of the compounds of the formula (I) can beexplained by their effects as ligands on adenosine A1 receptors and/oradenosine A2b receptors.

The present invention furthermore relates to medicaments which compriseat least one compound of the formula (I), preferably together with oneor more pharmacologically acceptable auxiliary substances or carriersubstances, and to their use for the abovementioned purposes.

All the customary administration forms, i.e. that is oral, parenteral,inhalatory, nasal, sublingual, rectal, local, such as in the case ofimplants or stents, or external, such as transdermal, are suitable foradministering the compounds of the formula (I). In the case ofparenteral administration, mention may be made, in particular, ofintravenous, intramuscular and subcutaneous administration, for exampleas a subcutaneous depot. Oral or parenteral administration is preferred.Oral administration is particularly preferred.

In this connection, the active compounds can be administered eitheralone or in the form of preparations. Suitable preparations for oraladministration include tablets, capsules, pellets, coated tablets,pills, granules, solid and liquid aerosols, syrups, emulsions,suspensions and solutions. In this connection, the active compound mustbe present in a quantity which is such that a therapeutic effect isachieved. In general, the active compound can be present in aconcentration of from 0.1 to 100% by weight, in particular of from 0.5to 90% by weight, preferably of from 5 to 80% by weight. In particular,the concentration of the active compound should be 0.5–90% by weight,i.e. the active compound should be present in quantities which aresufficient for achieving the stipulated dosage latitude.

For this purpose, the active compounds can be converted, in a mannerknown per se, into the customary preparations. This is effected usinginert, nontoxic, pharmaceutically suitable carrier substances,auxiliaries, solvents, vehicles, emulsifiers and/or dispersing agents.

Auxiliary substances which may be cited, by way of example, are: water,non-toxic organic solvents, such as paraffins, vegetable oils (e.g.sesame seed oil), alcohols (e.g. ethanol, glycerol), glycols (e.g.polyethylene glycol), solid carrier substances, such as natural orsynthetic mineral powders (e.g. talc or silicates), sugars (e.g.lactose), emulsifiers, dispersing agents (e.g. polyvinylpyrrolidone) andglidants (e.g. magnesium sulphate).

In the case of oral administration, tablets can naturally also containadditives, such as sodium citrate, together with admixed substances,such as starch, gelatin and the like. Furthermore, taste improvers ordyes can be added to aqueous preparations for oral administration.

In connection with parenteral administration, it has in general beenfound to be advantageous, for the purpose of achieving effectiveresults, to administer quantities of from about 0.1 to about 10 000μg/kg, preferably from about 1 to about 1 000 μg/kg, in particular fromabout 1 μg/kg to about 100 μg/kg of body weight. In the case of oraladministration, the quantity is from about 0.01 to about 10 mg/kg,preferably from about 0.05 to about 5 mg/kg, in particular from about0.1 to about 1 mg/kg of body weight.

Despite this, it can, where appropriate, be necessary to depart from theabovementioned quantities, depending on the body weight, the route ofadministration, the individual response to the active compound, thenature of the preparation and the time or interval at which theadministration takes place.

The present invention is illustrated by the following, non-limiting,preferred examples, which do not, however, restrict the invention in anyway.

Unless otherwise indicated, the percentage values in the followingexamples in each case refer to the weight; parts are parts by weight.

A. Assessing the Physiological Activity

I. Detecting the Cardiovascular Effect

After the thorax has been opened, the heart is rapidly removed fromanaesthetized rats and introduced into a conventional Langendorffapparatus. The coronary arteries are perfused at constant volume (10ml/min) and the perfusion pressure which arises in this connection isrecorded by way of an appropriate pressure sensor. A decrease in theperfusion pressure in this set-up corresponds to a relaxation of thecoronary arteries. At the same time, the pressure which the heartdevelops during each contraction is measured by way of a balloon, whichhas been introduced into the left ventricle, and a second pressuresensor. The frequency of the heart, which is beating in isolation, iscalculated from the number of contractions per unit time.

II. Determining the Adenosine A1, A2a, A2b and A3 Agonism

a) Determining the Adenosine Agonism Indirectly by Way of GeneExpression

Cells of the CHO (Chinese Hamster Ovary) permanent cell line aretransfected stably with the cDNA for the adenosine receptor subtypes A1,A2a and A2b. The adenosine A1 receptors are coupled to the adenylatecyclase by way of Gi proteins, while the adenosine A2a and A2b receptorsare coupled by way of Gs proteins. In correspondence with this, theformation of cAMP in the cell is inhibited or stimulated, respectively.After that, expression of the luciferase is modulated by way of acAMP-dependent promoter. The luciferase test is optimized, with the aimof high sensitivity and reproducibility, low variance and goodsuitability for implementation on a robot system, by varying severaltest parameters, such as cell density, duration of the growth phase andthe test incubation, forskolin concentration and medium composition. Thefollowing test protocol is used for pharmacologically characterizingcells and for the robot-assisted substance test screening:

The stock cultures are grown, at 37° C. and under 5% CO₂, in DMEM/F12medium containing 10% FCS (foetal calf serum) and in each case split1:10 after 2–3 days. The test cultures are seeded in 384-well plates atthe rate of from 1 000 to 3 000 cells per well and grown at 37° C. forapprox. 48 hours. The medium is then replaced with a physiologicalsodium chloride solution (130 mM sodium chloride, 5 mM potassiumchloride, 2 mM calcium chloride, 20 mM HEPES, 1 mM magnesium chloride6H₂O, 5 mM NaHCO₃, pH 7.4). The substances, which are dissolved in DMSO,are deleted 1:10 three times with this physiological sodium chloridesolution and pipetted into the test cultures (maximum finalconcentration of DMSO in the test mixture: 0.5%). In this way, finalsubstance concentrations of, for example, from 5 μM to 5 nM areobtained. 10 minutes later, forskolin is added to the A1 cells and allthe cultures are subsequently incubated at 37° C. for four hours. Afterthat, 35 μl of a solution which is composed of 50% lysis reagent (30 mMdisodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM TrisHCl,2 mM dithiothreitol (DTT), pH 7.8) and 50% luciferase substrate solution(2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mMmagnesium sulphate, 15 mM DTT, pH 7.8) are added to the test cultures,the plates are shaken for approx. 1 minute and the luciferase activityis measured using a camera system. The adenosine-analogous compound NECA(5-N-ethylcarboxamido-adenosine), which binds to all adenosine receptorsubtypes with high affinity and possesses an agonistic effect, is usedin these experiments as the reference compound (Klotz, K. N., Hessling,J., Hegler, J., Owman, C., Kull, B., Fredholm, B. B., Lohse, M. J.,Comparative pharmacology of human adenosine receptorsubtypes—characterization of stably transfected receptors in CHO cells,Naunyn Schmiedebergs Arch Pharmacol, 357 (1998), 1–9).

The following Table 1 gives the values which were obtained for thestimulation of different adenosine receptor subtypes by differentconcentrations of the compound from Example 1.

TABLE 1 Stimulation of adenosine receptors by different concentrationsof the compound from Example 1 Concentration of the compound fromExample 1 Receptor subtype 10 nmol 1 nmol 0.3 nmol A1 5 9 44 A2a 57 24 1A2b 88 64 29

The table gives the % values of the corresponding reference stimulus.The measured values for the A2a and A2b receptors are values in percentof the maximum stimulation achieved by NECA; the measured values for theA1 receptor are values in percent following direct prestimulation of theadenylate cyclase with 1μ molar forskolin (corresponds to the 100%value). A1 agonists accordingly exhibit a decrease in the activity ofthe luciferase (measured value less than 100%).

b) Determining the Adenosine Agonism Directly by way of Detecting cAMP

Cells of the CHO (Chinese Hamster Ovary) permanent cell line aretransfected stably with the cDNA for the adenosine receptor subtypes A1,A2a, A2b and A3. The binding of the substances to the A2a or A2breceptor subtypes is determined by measuring the intracellular cAMPcontent in these cells using a conventional radioimmunological assay(cAMP RIA, IBL GmbH, Hamburg, Germany).

When the substances act as agonists, the binding of the substances isexpressed as an increase in the intracellular content of cAMP. Theadenosine-analogous compound NECA (5-N-ethylcarboxamido-adenosine),which binds all adenosine receptor subtypes with high affinity andpossesses an agonistic effect, is used as the reference compound inthese experiments (Klotz, K. N., Hessling, J., Hegler, J., Owman, C.,Kull, B., Fredholm, B. B., Lohse, M. J., Comparative pharmacology ofhuman adenosine receptor subtypes—characterization of stably transfectedreceptors in CHO cells, Naunyn Schmiedebergs Arch Pharmacol, 357 (1998),1–9).

The adenosine receptors A1 and A3 are coupled to a Gi protein, i.e.stimulation of these receptors leads to inhibition of the adenylatecyclase and consequently to a lowering of the intracellular cAMP level.In order to identify A1/A3 receptor agonists, the adenylate cyclase isstimulated with forskolin. However, an additional stimulation of theA1/A3 receptors inhibits the adenylate cyclase, which means that A1/A3receptor agonists can be detected by a comparatively low content of cAMPin the cell.

In order to detect an antagonistic effect on adenosine receptors, therecombinant cells which are transfected with the corresponding receptorare prestimulated with NECA and the effect of the substances on reducingthe intracellular content of cAMP occasioned by this prestimulation isinvestigated. XAC (xanthine amine congener), which binds to alladenosine receptor subtypes with high affinity and possesses anantagonistic effect, is used as the reference compound in theseexperiments (Müller, C. E., Stein, B., Adenosine receptor antagonists:structures and potential therapeutic applications, CurrentPharmaceutical Design, 2 (1996) 501–530).

III. Pharmacokinetic Investigations

Pharmacokinetic data were determined after administering varioussubstances i.v. or p.o. as solutions to mice, rats and dogs. For this,blood samples were collected up to 24 hours after administration. Theconcentrations of the unaltered substance were determined bybioanalytical methods (HPLC or HPLC-MS) in the plasma samples which wereobtained from the blood samples. Pharmacokinetic parameters weresubsequently ascertained from the plasma concentration time courseswhich had been obtained in this way. The following Table 2 gives thebioavailability in the different species.

TABLE 2 Bioavailabilities following oral administration Mouse Rat DogCompound from not possible to not possible to 1.47% Example 22 indetermine* determine* (at 1 mg/ WO 00/125210 (at 3 mg/kg p.o.) (at 10mg/kg p.o.) kg p.o.) Compound from 22.1% 4.6% 48.2% Example 1 (at 1mg/kg p.o.) (at 1 mg/kg p.o.) (at 1 mg/ kg p.o.) *Plasma levels at allmeasurement time points were below the determination limit (<1 μg/l)B. Implementation Examples

EXAMPLE 1N-(4-{2-Amino-3,5-dicyano-6-[(2-pyridinylmethyl)sulphanyl]-4-pyridinyl}-phenyl)acetamide

1st Step:

N-[4-(2,2-Dicyanovinyl)phenyl]acetamide

311.4 g (1.9 mol) of 4-acetaminobenzaldehyde and 131 g (1.99 mol) ofmalonitrile are initially introduced in 1 330 ml of ethanol, and 6 ml ofpiperidine are then added. The mixture is stirred under reflux for 30minutes. After cooling down to room temperature, the crystals arefiltered off with suction and dried.

Yield: 318 g (79% of theory) Mass spectrum: sought-after relative molarmass: 211; found [M+H]⁺=212

2nd Step:

N-{4-[2-Amino-3,5-dicyano-6-(phenylsulphanyl)-4-pyridinyl]phenyl}acetamide

318 g (1.5 mol) of N-[4-(2,2-dicyanovinyl)phenyl]acetamide, 99 g (1.5mol) of malonitrile and 166 g (1.5 mol) of thiophenol are initiallyintroduced in 2 000 ml of ethanol, and 6.7 ml of triethylamine are thenadded. The mixture is stirred under reflux for 2 hours, in connectionwith which crystallization takes place. After the mixture has cooleddown to room temperature, the product is filtered off with suction anddried in vacuo.

Yield: 170.3 g (29% of theory) Mass spectrum: sought-after relativemolar mass: 385; found [M+H]⁺=386

3rd Step:

N-[4-(2-Amino-3,5-dicyano-6-sulphanyl-4-pyridinyl)phenyl]acetamide

30.83 g (80 mmol) ofN-{4-[2-amino-3,5-dicyano-6-(phenylsulphanyl)-4-pyridinyl]-phenyl}-acetamideare dissolved in 120 ml of DMF under argon, after which 9.36 g (120mmol) of sodium sulphide are added and the mixture is stirred at 80° C.for 2 hours. A solution of 20 ml of 1N aqueous HCl in 44 ml of water isthen added dropwise at from 40 to 65° C., after which the crystals whichhave formed during this procedure are filtered off with suction andwashed with water. The precipitate is suspended in 200 ml of methanoland stirred under reflux for 5 minutes. After cooling down to roomtemperature, the precipitate is filtered off with suction, washed withmethanol and diethyl ether and dried in vacuo.

Yield: 24.5 g (88% of theory) Mass spectrum: sought-after relative molarmass: 309; found [M+H]⁺=310.1

4th Step:

N-(4-{2-Amino-3,5-dicyano-6-[(2-pyridinylmethyl)sulphanyl]-4-pyridinyl}-phenyl)acetamide

9.28 g (30 mmol) ofN-[4-(2-amino-3,5-dicyano-6-sulphanyl-4-pyridinyl)phenyl]-acetamide,7.38 g (45 mmol) of 2-picolyl chloride hydrochloride and 10.08 g (120mmol) of sodium hydrogen carbonate are stirred at room temperature in100 ml of DMF. After 2 hours, 100 ml of water are added dropwise at from40 to 50° C.

After cooling down to room temperature, the yellow-orange crystals arefiltered off with suction and dried in vacuo.

Yield: 10.42 g (86% of theory) Mass spectrum: sought-after relativemolar mass: 400; found [M+H]⁺=401 ¹H NMR (300 MHz, DMSO-d₆): δ=2.1 (s,3H), 4.6 s (2H), 7.4 (dd, 1H), 7.45 (d, 1H), 7.65 (d, 2H), 7.75 (m, 3H),8.1 (s broad, 2H), 8.5 (d, 1H), 10.25 (s, 1H).

EXAMPLE 2 Methyl4-(2-amino-3,5-dicyano-6-{[(2-methyl-1,3-thiazol-4-yl)methyl]-sulphanyl}-4-pyridinyl)phenylcarbamate

1st Step:

Methyl 4-(2-amino-3,5-dicyano-6-sulphanyl-4-pyridinyl)phenylcarbamate

8.5 g (47.4 mmol) of methyl 4-formylphenylcarbamate (Witek et al,Journal f. Prakt. Chemie 321, 804–812 (1979)), 9.5 g (94.9 mmol) ofcyanothioacetamide and 9.6 g (94.88 mmol) of N-methylmorpholine areheated under reflux in ethanol for 3 hours. After evaporation,dichloromethane/methanol is added to the residue and the whole isfiltered. After having been absorbed to kieselguhr the filtrate ispurified by chromatography on silica gel (eluent:dichloromethane/methanol, 100:2 to 100:6). The product fractions arecombined and evaporated. The evaporation residue is dissolved in 200 mlof 1N aqueous sodium hydroxide solution and the whole is then filtered.300 ml of 1N aqueous hydrochloric acid are added to the filtrate and theresulting precipitate is filtered off with suction and dried in vacuo.

Yield: 2.7 g (17% of theory) Mass spectrum: sought-after relative molarmass: 325; found [M+H]⁺=326 ¹H NMR (200 MHz, DMSO-d₆): δ=3.7 s (3H), 7.4(d, 2H), 7.6 (d, 2H), 8.1 (s broad, 2H), 10.0 (s, 1H)

2nd Step:

Methyl4-(2-amino-3,5-dicyano-6-{[(2-methyl-1,3-thiazol-4-yl)methyl]-sulphanyl}-4-pyridinyl)phenylcarbamate

32.5 mg (0.1 mmol) of methyl4-(2-amino-3,5-dicyano-6-sulphanyl-4-pyridinyl)-phenylcarbamate and 27.6mg (0.15 mmol) of 4-(chloromethyl)-2-methyl-1,3-thiazole hydrochlorideare shaken overnight in 0.4 ml of DMF together with 33.6 mg (0.4 mmol)of sodium hydrogen carbonate. The reaction mixture is filtered andpurified by preparative HPLC.

Column: Nucleosil 5C18 Nautilus, 5 μm, 20×50 mm,

Precolumn: Gromsil ODS 4 HE 15 μm 10×20 mm.

Flow rate: 25 ml/min.

Gradient (A=acetonitrile, B=water+0.3% trifluoroacetic acid):

  0 min 10% A;   2 min 10% A;   6 min 90% A; 7.00 min 90% A; 7.10 min10% A;   8 min 10% A.

Detection: 220 nm. Injection volume: 600 μl

The product fraction is evaporated in vacuo.

Yield: 15.8 mg (36% of theory)

Mass spectrum: sought-after relative molar mass: 436; found [M+H]⁺=437

Abbreviations employed:

DMF Dimethylformamide DMSO Dimethylsulphoxide HEPES2-[4-(2-Hydroxyethyl)piperazino]ethanesulphonic acid HPLC High pressureor high performance liquid chromatography NMR Nuclear magnetic resonancespectroscopy RT Room temperature Tris2-Amino-2-(hydroxymethyl)-1,3-propanediol

1. A compound of the formula (I)

in which R¹ denotes (C₁–C₄)-alkyl, (C₁–C₄)-alkoxy, mono- ordi-(C₁–C₄)-alkylamino, and R² denotes pyridyl or thiazolyl, whichradicals can be substituted by halogen, amino or (C₁–C₄)-alkyl, or asalt, hydrate, or hydrate of the salt thereof.
 2. The compound of theformula (I) according to claim 1, in which R¹ denotes methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl, and R²denotes 2-pyridyl, thiazol-4-yl or thiazol-5-yl, which radicals can besubstituted by chlorine, amino or methyl, or a salt, hydrate, or hydrateof the salt, thereof.
 3. The compound according to claim 1 or claim 2having the following structure

or a salt, hydrate, or hydrate of the salt, thereof.
 4. A process forpreparing a compound of the formula (I)

in which R¹ denotes (C₁–C₄)-alkyl, (C₁–C₄)-alkoxy, mono- ordi-(C₁–C₄)-alkylamino, and R² denotes pyridyl or thiazolyl, whichradicals can be substituted by halogen, amino or (C₁–C₄)-alkyl, whereina compound of the formula (II)

in which R¹ has the meaning given above, is reacted with a compound ofthe formula (III)R²—CH₂—X  (III) in which R² has the meaning given above and X representsa leaving group.
 5. A pharmaceutical composition comprising one or morecompounds of the formula (I), according to claim 1, and at least onepharmaceutically acceptable carrier.
 6. A method for treatment ofhypertension comprising administering an effective amount of a compoundof claim 1.